Modification of genomic DNA is of immense importance for basic and applied research. Genomic modifications have the potential to elucidate and in some cases to cure the causes of disease and to provide desirable traits in the cells and/or individuals comprising said modifications. Genomic modification may include, for example, modification of plant, animal, fungal, and/or prokaryotic genomic modification. One area in which genomic modification is practiced is in the modification of plant genomic DNA.
Modification of plant genomic DNA is of immense importance to both basic and applied plant research. Transgenic plants with stably modified genomic DNA can have new traits such as herbicide tolerance, insect resistance, and/or accumulation of valuable proteins including pharmaceutical proteins and industrial enzymes imparted to them. The expression of native plant genes may be up- or down-regulated or otherwise altered (e.g., by changing the tissue(s) in which native plant genes are expressed), their expression may be abolished entirely, DNA sequences may be altered (e.g., through point mutations, insertions, or deletions), or new non-native genes may be inserted into a plant genome to impart new traits to the plant.
The most common methods for modifying plant genomic DNA tend to modify the DNA at random sites within the genome. Such methods include, for example, Agrobacterium-mediated plant transformation and biolistic transformation, also referred to as particle bombardment. In many cases, however, it is desirable to modify the genomic DNA at a pre-determined target site in the plant genome of interest, e.g., to avoid disruption of native plant genes or to insert a transgene cassette at a genomic locus that is known to provide robust gene expression. Only recently have technologies for targeted modification of plant genomic DNA become available. Such technologies rely on the creation of a double-stranded break (DSB) at the desired site. This DSB causes the recruitment of the plant's native DNA-repair machinery to the DSB. The DNA-repair machinery may be harnessed to insert heterologous DNA at a pre-determined site, to delete native plant genomic DNA, or to produce point mutations, insertions, or deletions at a desired site.